Methods and compositions for treating autoimmune disease

ABSTRACT

The disclosure relates to OX-2/CD200 (herein referred to as CD200) antibodies and methods of treating autoimmune disease.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/962,044, filed Jul. 25, 2007, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The disclosure relates to OX-2/CD200 (herein referred to as CD200)antibodies and methods of treating autoimmune disease.

BACKGROUND

Autoimmunity is the failure of an organism to recognize its ownconstituent parts (down to the sub-molecular levels) as “self”, whichresults in an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune disease. In order to inhibit harmful immune reactions in suchinstances, immunosuppressive agents such as corticosteroids and cytokineantagonists may be administered to patients. However these generalimmunosuppressives can elicit undesirable side effects includingtoxicity and reduced resistance to infection. Thus alternative, andperhaps more specific, methods of treating autoimmunity are needed.

Several immunomodulatory therapies, including antibody therapies, haveproven successful in the treatment of certain autoimmune disorders.However there is a clinical need for additional antibody therapies forthe treatment of autoimmune disorders. Furthermore, there is a relatedneed for humanized or other chimeric human/mouse monoclonal antibodies.In well publicized studies, patients administered murine anti-TNF (tumornecrosis factor) monoclonal antibodies developed anti-murine antibodyresponses to the administered antibody (Exley A. R., et al., Lancet335:1275-1277 (1990)). This type of immune response to the treatmentregimen, commonly referred to as the human anti-mouse antibody (HAMA)response (Mirick et al. Q J Nucl Med Mol Imaging 2004; 48: 251-7),decreases the effectiveness of the treatment and may even render thetreatment completely ineffective. Humanized or chimeric human/mousemonoclonal antibodies have been shown to significantly decrease the HAMAresponse and to increase the therapeutic effectiveness of antibodytreatments. See, for example, LoBuglio et al., Proc. Natl. Acad. Sci.USA 86:4220-4224 (June 1989). Furthermore, antibodies in whichparticular functionalities are either enhanced or reduced may finduseful applications in the clinic.

CD200, a molecule expressed on the surface of numerous cell typesincluding B cells, some T cells and dendritic cells and other cells,which possesses a high degree of homology to molecules of theimmunoglobulin gene family, has previously been thought to be implicatedin immune suppression (Gorczynski et al., Transplantation 65:1106-1114(1998)). The prior art appears to show, for example, thatCD200-expressing cells can inhibit the stimulation of Th1 cytokineproduction.

SUMMARY

In certain aspects the disclosure provides a method for treating apatient with an autoimmune disease, said method comprising administeringa therapeutically effective amount of an anti-CD200 antibody orantigen-binding fragment thereof to said patient. In certainembodiments, said autoimmune disease is selected from the groupincluding but not limited to rheumatoid arthritis, inflammatory boweldisease, systemic lupus erythematosus, multiple sclerosis, Hashimoto'sthyroiditis, pernicious anemia, Addison's disease, type I diabetes,dermatomyositis, Sjogren's syndrome, lupus erythematosus, myastheniagravis, Reiter's syndrome, idiopathic thrombocytopenic purpura,hemolytic anemia, Wegener's granulomatosis, refractory dermatomyositis,cold agglutinin disease associated with indolent lymphoma, acquiredfactor VIII inhibitors disease and Grave's disease.

In certain embodiments, said antibody or antigen-binding fragmentthereof blocks the production of auto-antibodies. In certainembodiments, said auto-antibodies are selected from IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgA, IgD, and/or IgE immunoglobulins. In certainembodiments, said antibody or antigen-binding fragment thereof does notblock the production of auto-antibodies.

In certain embodiments, said antibody or antigen-binding fragmentthereof is an antagonistic antibody. In certain embodiments, saidantibody or antigen-binding fragment thereof is an agonistic antibody.

In certain embodiments, said antibody or antigen-binding fragmentthereof modulates expression of cytokines in said patient. In certainembodiments, said antibody or antigen-binding fragment thereof enhancesproduction of a cytokine in said patient selected from the groupconsisting of: IL-12, IL-10 and IL-4.

In certain embodiments, said antibody or antibody fragment thereof isselected from the group consisting of a polyclonal antibody, amonoclonal antibody or antibody fragment thereof, a recombinantantibody, a diabody, a chimerized or chimeric antibody or antibodyfragment thereof, a humanized antibody or antibody fragment thereof, adeimmunized human antibody or antibody fragment thereof, a fully humanantibody or antibody fragment thereof, a single chain antibody, an Fv,an Fd, an Fab, an Fab′, and an F(ab′)₂. In certain embodiments, saidantibody is a monoclonal antibody. In certain embodiments, saidanti-CD200 antibody or antibody fragment thereof is conjugated to amolecule selected from the group consisting of a polymer and apolypeptide. In certain embodiments, said polymer is poly(ethylene)glycol.

In certain embodiments, said antibody or antigen-binding fragmentthereof is administered for at least one month to said mammal. Incertain embodiments, said antibody or antigen-binding fragment thereofis administered for at least one year to said mammal. In certainembodiments, said antibody or antigen-binding fragment thereof isadministered chronically to said mammal.

In certain embodiments, said antibody or antigen-binding fragmentthereof is administered systemically to said mammal. In certainembodiments, said antibody or antigen-binding fragment thereof isadministered locally to said mammal.

In certain embodiments, the methods of the disclosure further compriseadministering a second agent or therapy. In certain embodiments, thesecond agent comprises one or more of the following characteristics: a)regulatory activity on T cells; and b) immunomodulatory activity. Incertain embodiments, said second agent or therapy is selected from thegroup consisting of an immunosuppressive agent, immunomodulatory agent,heteroclitic peptide, antibody, antigen-binding fragment, nucleic acid,small molecule, organometallic compound, polypeptide, aptamer,spiegelmer, chemical, inorganic compound, metal, prodrug, andpeptidomimetic compound. In certain embodiments, the immunomodulatory orimmunosuppressive agent is a calcineurin inhibitor. In certainembodiments, the calcineurin inhibitor is selected from tacrolimus(FK-506) and cyclosporine A. In certain embodiments, theimmunomodulatory or immunosuppressive agent is selected from the groupconsisting of adriamycin, azathiopurine, busulfan, cyclophosphamide,cyclosporine A, Cytoxan, fludarabine, 5-fluorouracil, methotrexate,mycophenolate mofetil, a nonsteroidal anti-inflammatory, sirolimus(rapamycin), and tacrolimus (FK-506). In certain embodiments, theimmunomodulatory or immunosuppressive agent is an antibody selected fromthe group consisting of muromonab-CD3, alemtuzumab, basiliximab,daclizumab, rituximab, IVIg and anti-thymocyte globulin. In certainembodiments, said second agent is administered either sequentially orsimultaneously.

The invention contemplates combinations of any of the foregoing aspectsand embodiments of the invention. Other embodiments are described in thedescription. All references cited herein are hereby incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows anti-CD200 treatment reduces the severity of collageninduced arthritis. DBA/1LacJ mice were administered 5 mg/kg dose ofeither anti-CD200 or isotype-matched control mAb by i.p. injection fromday 1 to day 7 and day 21 to day 25 after initial BCII immunization onday 1. Data are presented as mean±SEM.

FIG. 2 shows anti-CD200 treatment blocks the production of anti-collagenantibody production. Serum levels and subtypes of anti-BCII Abs wereevaluated for the indicated treatment groups. Data are presented asmean±SEM.

FIGS. 3A-3B show anti-CD200 treatment can ameliorate established jointinflammation independent of the effect on autoantibody production. A)DBA/1LacJ mice were administered 5 mg/kg dose of either anti-CD200 orisotype-matched control mAb by i.p. injection on day 21 to day 30 afterinitial BCII immunization on day 1. B) Serum levels and subtypes ofanti-BCII Abs were evaluated for the indicated treatment groups. Dataare presented as mean±SEM.

FIGS. 4A-4B show anti-CD200 treatment affects splenic cytokine profileswhen administered at various time points relative to collagenimmunization of DBA/1 mice. A) Spleen cells were isolated from BCIIimmunized DBA/1LacJ mice, which were treated with either anti-CD200 orisotype-matched control mAb from day 1 to day 7 and day 21 to day25after initial BCII immunization on day1. B) Spleen cells were isolatedfrom BCII immunized DBA/1LacJ mice, which were treated with eitheranti-CD200 or isotype-matched control mAb from day 21 to day30 afterinitial BCII immunization on day1. Data are presented as mean±SEM.

FIG. 5 shows the effect of alteration of cytokine profile afteranti-CD200 treatment in an allogenic immune response, where BALB/c micewere immunized with C57B/c spleen cells. Data are presented as mean±SEM.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS I. Overview

Prior art, especially numerous articles by Gorczynski, has seemed toindicate that the molecule CD200 is immunosuppressive. For example,Gorczynski et al., Clin. Immunol. 104:256-264 (2002) teaches that in acollagen-induced arthritis (CIA) model in mice treatment with CD200 (ina CD200Fc form) ameliorates CIA. They state that work has shown thatCD200 binds its receptor and the immunosuppressive activity is via thereceptor. The paper teaches that use of an anti-CD200 receptor antibodythat crosslinks the receptor has this same activity of ameliorating CIA,the antibody apparently being an agonistic antibody. It is likely thatCD200, which is a member of the Ig superfamily, acts similarly to anantibody and acts to crosslink the CD200 receptor thereby activating thereceptor which in turn results in immunosuppression.

Based on the prior art, one would expect that treatment of an animal orpatient with an anti-CD200 antibody should result in an enhanced immuneresponse. The logic is that antibodies to CD200 would bind the CD200thereby preventing CD200 from binding to its receptor. Without aCD200:CD200 receptor interaction the CD200 receptor would not beactivated and there would be no immune suppression, thereby resulting inenhanced inflammation or autoimmune effect. For diseases such as CIAthis would mean that treatment with anti-CD200 should exacerbate thedisease.

II CD200 Antibodies

CD200 is a highly conserved type I transmembrane glycoprotein expressedon various cell types including cells of the immune system (e.g.,T-cells, B-cells, and dendritic cells (Barclay et al., 2002 TRENDSImmunol. 23:285-290)). The protein interacts with its receptor CD200R onmyeloid cells and sub-populations of T cells (Wright et al. J. Immunol.2003 171: 3034-3046 and Wright et al., Immunity 2000 13:233-242); theCD200:CD200R interaction has been thought to deliver an immunomodulatorysignal to cells and induce immunosuppression includingapoptosis-associated immune tolerance (Rosenblum et al. 2004 Blood 103:2691-2698). Thus it has been thought that agents that modulate thefunction or activity of CD200 and/or its receptor may result in enhancedimmunosuppressive effects. In addition, agents that specifically bindCD200 (but that may or may not modulate the CD200:CD200R interaction)may trigger downstream events that modulate the effects of CD200.

In certain aspects, the present disclosure relates to CD200 modifiers.As used herein, the term modifier includes any agent that is capable ofmodulating the activity, function and/or the expression of CD200 or itsreceptor. Examples include but are not limited to polypeptides,antibodies, small molecules, aptamers, spiegelmers, locked nucleic acid(LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, nucleic acidconstructs (e.g., gene-targeting constructs, antisense constructs, RNAinterference (RNAi) constructs, etc.) and peptidomimetics. In certainembodiments, the antibody disrupts the interaction of CD200 and CD200R.In other embodiments, the CD200 antibodies are capable of increasing theimmunosuppressive effects of CD200 or are capable of targetingCD200-expressing cells for depletion or elimination.

In certain aspects, the CD200 modifiers are polypeptides. Polypeptidesutilized in the present disclosure can be constructed using differenttechniques which are known to those skilled in the art. In oneembodiment, the polypeptides are obtained by chemical synthesis. Inother embodiments, the polypeptides are constructed from a fragment orseveral fragments. In further embodiments, the polypeptide is ananti-CD200 antibody as described herein.

As used herein, the term “antibodies” refers to complete antibodies orantibody fragments capable of binding to CD200 or CD200R. Included areFab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal and polyclonal antibodies,engineered antibodies (including chimeric, single chain, CDR-grafted,humanized, fully human antibodies, and artificially selectedantibodies), and synthetic or semi-synthetic antibodies produced usingphage display or alternative techniques. Also included are antibodiesengineered or produced in ways to contain variant or altered constant orFc regions with either increased or decreased ability to bind one ormore effector cells; such variant antibodies include but are not limitedto antibodies in which the constant or Fc region contains alteredglycosylation patterns. Small fragments, such as Fv and scFv, possessadvantageous properties for diagnostic and therapeutic applications onaccount of their small size and consequent superior tissue distribution.Antibodies with engineered or variant constant or Fc regions can beuseful in modulating effector functions, such as, for example, ADCC andCDC.

Such antibodies with engineered or variant constant or Fc regions may beuseful in instances where CD200 is expressed in normal tissue, forexample; variant anti-CD200 antibodies without effector function inthese instances may elicit the desired therapeutic response while notdamaging normal tissue. Furthermore, antibodies, variant antibodies, andfragments thereof may be blocking (i.e., the antibodies or fragmentsinhibit the interaction of CD200 and CD200R) or agonistic (i.e., theantibodies or fragments enhance the interaction of CD200 and CD200R).

The disclosure also relates to anti-CD200 antibodies comprising heavyand light chains as provided herein, including heavy and light chainsthat are homologous or similar to the heavy and/or light chains providedherein. “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position;when the equivalent site occupied by the same or a similar amino acidresidue (e.g., similar in steric and/or electronic nature), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of identical or similar amino acids atpositions shared by the compared sequences. The term “homology”describes a mathematically based comparison of sequence similaritieswhich is used to identify genes or proteins with similar functions ormotifs. As used herein, “identity” means the percentage of identicalnucleotide or amino acid residues at corresponding positions in two ormore sequences when the sequences are aligned to maximize sequencematching, i.e., taking into account gaps and insertions. Thus methods todetermine identity are designed to give the largest match between thesequences tested (see Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988), Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984), BLASTP, BLASTN, FASTA (Altschul, S. F. et al., J. Mol. Biol.215: 403-410 (1990) and Altschul et al. Nucleic Acids Res. 25: 3389-3402(1997)) and BLAST X (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990)). A sequence which is “unrelated” or “non-homologous” shares lessthan 40% identity, though preferably less than 25% identity with asequence of the present disclosure. In comparing two sequences, theabsence of residues (amino acids or nucleic acids) or presence of extraresidues also decreases the identity and homology/similarity.

Accordingly, the disclosure relates to antibodies as described hereinwithout the leader sequences. Thus antibodies of the disclosure maycomprise heavy and light chains (as described herein) in which theleader sequence is either absent or replaced by a different leadersequence. If host cells are used to produce antibodies of the presentdisclosure, appropriate leader sequences may therefore be selectedaccording to the particular host cell used.

Antibodies may be produced by methods well known in the art. Forexample, monoclonal anti-CD200 antibodies may be generated using CD200positive cells, CD200 polypeptide, or a fragment of CD200 polypeptide,as an immunogen, thus raising an immune response in animals from whichantibody-producing cells and in turn monoclonal antibodies may beisolated. The sequence of such antibodies may be determined and theantibodies or variants thereof produced by recombinant techniques.Recombinant techniques may be used to produce chimeric, CDR-grafted,humanized and fully human antibodies based on the sequence of themonoclonal antibodies as well as polypeptides capable of binding toCD200.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using CD200-positive cells, or polypeptidesderived therefrom, as bait to isolate the antibodies or polypeptides onthe basis of target specificity. The production and isolation ofnon-human and chimeric anti-CD200 antibodies are well within the purviewof the skilled artisan.

Recombinant DNA technology is used to improve the antibodies produced innon-human cells. Thus, chimeric antibodies may be constructed in orderto decrease the immunogenicity thereof in diagnostic or therapeuticapplications. Moreover, immunogenicity may be minimized by humanizingthe antibodies by CDR grafting and, optionally, framework modification.See, U.S. Pat. No. 5,225,539, the contents of which are incorporatedherein by reference.

Antibodies may be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic, for example bicistronic, DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, for exampleWO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith,1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith, 1988, Gene 73,pp 305-318; De La Cruz et al., 1988, Journal of Biological Chemistry,263 pp 4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409;WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No.5,571,698; U.S. Pat. No. 6,040,136; Davis et al., 1999, CancerMetastasis Rev., 18(4):421-5; Taylor, et al., Nucleic Acids Research 20(1992): 6287-6295; Tomizuka et al., Proc. Natl. Academy of Sciences USA97(2) (2000): 722-727. The contents of all these references areincorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of CD200-positive cells, byimmunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with one or more surface polypeptides derived from aCD200-positive cell line, or with Protein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against CD200 in a suitablemammal. For example a rabbit is immunized with pooled samples fromCD200-positive tissue or cells or CD200 polypeptide or fragmentsthereof. A phage display library produced from the immunized rabbit isconstructed and panned for the desired antibodies in accordance withmethods well known in the art (such as, for example, the methodsdisclosed in the various references incorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beexpanded from deep-frozen cultures by thawing and propagation in vitroor as ascites in vivo.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against CD200. Inthat process, a suitable mammal, for example a Balb/c mouse, isimmunized with one or more polypeptides or antigenic fragments of CD200or with one or more polypeptides or antigenic fragments derived from aCD200-positive cell, the CD200-positive cell itself, or an antigeniccarrier containing a purified polypeptide as described.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with a CD200-positive Chronic Lymphocytic Leukemia (CLL)cell line are fused with cells of the myeloma cell line PAI or themyeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are thenscreened for secretion of the desired antibodies and positive hybridomacells are cloned.

Preferred is a process for the preparation of a hybridoma cell line,characterized in that Balb/c mice are immunized by injectingsubcutaneously and/or intraperitoneally between 10⁶ and 10⁷ cells of aCD200-positive cell line several times, e.g. four to six times, overseveral months, e.g. between two and four months. Spleen cells from theimmunized mice are taken two to four days after the last injection andfused with cells of the myeloma cell line PAI in the presence of afusion promoter, preferably polyethylene glycol. Preferably, the myelomacells are fused with a three- to twenty-fold excess of spleen cells fromthe immunized mice in a solution containing about 30% to about 50%polyethylene glycol of a molecular weight around 4000. After the fusion,the cells are expanded in suitable culture media as describedhereinbefore, supplemented with a selection medium, for example HATmedium, at regular intervals in order to prevent normal myeloma cellsfrom overgrowing the desired hybridoma cells.

The antibodies and fragments thereof can be “chimeric”. Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced into human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication.

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Also see Staelens etal. 2006 Mol Immunol 43: 1243-1257. In particular embodiments, humanizedforms of non-human (e.g., mouse) antibodies are human antibodies(recipient antibody) in which hypervariable (CDR) region residues of therecipient antibody are replaced by hypervariable region residues from anon-human species (donor antibody) such as a mouse, rat, rabbit, ornon-human primate having the desired specificity, affinity, and bindingcapacity. In some instances, framework region residues of the humanimmunoglobulin are also replaced by corresponding non-human residues (socalled “back mutations”). In addition, phage display libraries can beused to vary amino acids at chosen positions within the antibodysequence. The properties of a humanized antibody are also affected bythe choice of the human framework. Furthermore, humanized and chimerizedantibodies can be modified to comprise residues that are not found inthe recipient antibody or in the donor antibody in order to furtherimprove antibody properties, such as, for example, affinity or effectorfunction.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992). Transgenic mice strains canbe engineered to contain gene sequences from unrearranged humanimmunoglobulin genes. The human sequences may code for both the heavyand light chains of human antibodies and would function correctly in themice, undergoing rearrangement to provide a wide antibody repertoiresimilar to that in humans. The transgenic mice can be immunized with thetarget protein (e.g., CD200, fragments thereof, or cells expressingCD200) to create a diverse array of specific antibodies and theirencoding RNA. Nucleic acids encoding the antibody chain components ofsuch antibodies may then be cloned from the animal into a displayvector. Typically, separate populations of nucleic acids encoding heavyand light chain sequences are cloned, and the separate populations thenrecombined on insertion into the vector, such that any given copy of thevector receives a random combination of a heavy and a light chain. Thevector is designed to express antibody chains so that they can beassembled and displayed on the outer surface of a display packagecontaining the vector. For example, antibody chains can be expressed asfusion proteins with a phage coat protein from the outer surface of thephage. Thereafter, display packages can be screened for display ofantibodies binding to a target.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)). Synthetic phage libraries can be created which userandomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See U.S. Pat. Nos. 6,794,132,6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma 2:361-367,the contents of which are incorporated by reference.

For the generation of human antibodies, also see Mendez et al. NatureGenetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med.188:483-495 (1998), the disclosures of which are hereby incorporated byreference. Human antibodies are further discussed and delineated in U.S.Pat. Nos. 5,939,598 and 6,673,986. Also see U.S. Pat. Nos. 6,114,598,6,075,181, and 6,162,963, all filed Jun. 5, 1995. Also see U.S. Pat. No.6,150,584, filed Oct. 2, 1996 and U.S. Pat. Nos. 6,713,610 and 6,657,103as well as U.S. patent application Ser. Nos. 10/421,011 (US 2003-0229905A1), 10/455,013 (US 2004-0010810 A1), 10/627,250 (US 2004-0093622 A 1),10/656,623 (US 2006-0040363 A1), 10/658,521 (US 2005-0054055 A1),10/917,703 (US 2005-0076395 A1) and 10/978,297 (US 2005-0287630 A1). Seealso PCT/US93/06926 filed on Jul. 23, 1993, European Patent No. EP 0 463151 B1, grant published Jun. 12, 1996, International Patent ApplicationNo. WO 94/02602, published Feb. 3, 1994, International PatentApplication No. WO 96/34096, published Oct. 31, 1996, and WO 98/24893,published Jun. 11, 1998. The disclosures of each of the above-citedpatents, applications, and references are hereby incorporated byreference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat. No.5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn,and GenPharm International. Also see U.S. Pat. Nos. 5,569,825,5,877,397, 6,300,129, 5,874,299, 6,255,458, and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Application Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al. (1992 Nucleic Acids Res., 20: 6287),Chen et al. (1993 Int. Immunol. 5: 647), Tuaillon et al. (1993 Proc.Natl. Acad. Sci. USA. 90: 3720-4), Choi et al., (1993 Nature Genetics 4:117), Lonberg et al. (1994 Nature 368: 856-859), Taylor et al. (1994International Immunology 6: 579-591), and Tuaillon et al. (1995 J.Immunol. 154: 6453-65), Fishwild et al. (1996 Nature Biotechnology 14:845), and Tuaillon et al. (2000 Eur. J. Immunol. 10: 2998-3005), thedisclosures of which are hereby incorporated by reference in theirentirety.

In certain embodiments, de-immunized anti-CD200 antibodies orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof may be modified so as to render theantibody or antigen-binding fragment thereof non-immunogenic, or lessimmunogenic, to a given species. De-immunization can be achieved bymodifying the antibody or antigen-binding fragment thereof utilizing anyof a variety of techniques known to those skilled in the art (see e.g.,PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, anantibody or antigen-binding fragment thereof may be de-immunized byidentifying potential T cell epitopes and/or B cell epitopes within theamino acid sequence of the antibody or antigen-binding fragment thereofand removing one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized anti-CD200antibodies described herein include de-immunized antigen-bindingfragments, Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies,murine antibodies, engineered antibodies (such as, for example,chimeric, single chain, CDR-grafted, humanized, fully human antibodies,and artificially selected antibodies), synthetic antibodies andsemi-synthetic antibodies.

In a further embodiment, recombinant DNA comprising an insert coding fora heavy chain variable domain and/or for a light chain variable domainof antibodies directed to CD200 or a CD200-positive cell line areproduced. The term DNA includes coding single stranded DNAs, doublestranded DNAs consisting of said coding DNAs and of complementary DNAsthereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or a lightchain variable domain of antibodies directed to CD200 or theCD200-positive cell line can be enzymatically or chemically synthesizedDNA having the authentic DNA sequence coding for a heavy chain variabledomain and/or for the light chain variable domain, or a mutant thereof.A mutant of the authentic DNA is a DNA encoding a heavy chain variabledomain and/or a light chain variable domain of the above-mentionedantibodies in which one or more amino acids are deleted, inserted, orexchanged with one or more other amino acids. Preferably saidmodification(s) are outside the CDRs of the heavy chain variable domainand/or of the light chain variable domain of the antibody inhumanization and expression optimization applications. The term mutantDNA also embraces silent mutants wherein one or more nucleotides arereplaced by other nucleotides with the new codons coding for the sameamino acid(s). The term mutant sequence also includes a degeneratesequence. Degenerate sequences are degenerate within the meaning of thegenetic code in that an unlimited number of nucleotides are replaced byother nucleotides without resulting in a change of the amino acidsequence originally encoded. Such degenerate sequences may be useful dueto their different restriction sites and/or frequency of particularcodons which are preferred by the specific host, particularly E. coli,to obtain an optimal expression of the heavy chain murine variabledomain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an antibody directed to CD200 or a CD200-positivecell line fused to a human constant domain IgG, for example γ1, γ2, γ3or γ4, in particular embodiments γ1 or γ4, may be used. Recombinant DNAsincluding an insert coding for a light chain murine variable domain ofan antibody fused to a human constant domain κ or λ, preferably κ, arealso provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent. The DNA coding for an agent is intended to be aDNA coding for the agent useful in diagnostic or therapeuticapplications. Thus, agent molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an agent has the sequenceof a naturally occurring enzyme or toxin encoding DNA, or a mutantthereof, and can be prepared by methods well known in the art.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label. Alternatively, the monoclonal antibody orantigen-binding fragment can be conjugated to an agent such as, forexample, a cytotoxic agent, a small molecule, a hormone, an enzyme, agrowth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, aprodrug, a nucleic acid molecule including coding sequences (such asantisense, RNAi, gene-targeting constructs, etc.), or a detectable label(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.).In certain embodiments, an anti-CD200 polypeptide or antigen-bindingfragment (e.g., Fab, Fv, single-chain scFv, Fab′ and F(ab′)₂) is linkedto a molecule that increases the half-life of the polypeptide orantigen-binding fragment. Molecules that may be linked to saidanti-CD200 polypeptide or antigen-binding fragment include but are notlimited to serum proteins including albumin, polypeptides, otherproteins or protein domains, and PEG.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan, R. C. and Berg, P., Proc. Natl. Acad. Sci., USA, 78:2072 (1981)) or Tn5 neo (Southern, P. J. and Berg, P., J. Mol. Appl.Genet., 1: 327 (1982)). The selectable marker gene can be either linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection (Wigler, M. et al., Cell, 16: 77 (1979)). Asecond class of vectors utilizes DNA elements which confer autonomouslyreplicating capabilities to an extrachromosomal plasmid. These vectorscan be derived from animal viruses, such as bovine papillomavirus(Sarver, N. et al., Proc. Natl. Acad. Sci., USA, 79: 7147 (1982)),polyoma virus (Deans, R. J. et al., Proc. Natl. Acad. Sci., USA, 81:1292 (1984)), or SV40 virus (Lusky, M. and Botchan, M., Nature, 293: 79(1981)).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, H. andBerg, P., Mol. Cell. Biol., 3: 280 (1983); Cepko, C. L. et al., Cell,37: 1053 (1984); and Kaufman, R. J., Proc. Natl. Acad. Sci., USA, 82:689 (1985).

In certain embodiments, an anti-CD200 antibody may be a blocking oragonistic. As used herein, a blocking antibody is one that blocks theinteraction between CD200 and CD200R. An agonistic antibody is one thatenhances the interaction between CD200 and CD200R. Thus in certainembodiments, an anti-CD200 antibody is either a blocking or agonisticmurine, chimeric, humanized, human or de-immunized antibody.

The CD200 antibodies and polypeptides and/or antibodies utilized in thepresent disclosure are especially indicated for diagnostic andtherapeutic applications as described herein. Accordingly CD200antibodies and anti-CD200 antibodies and variants thereof may be used intherapies, including combination therapies, in the diagnosis andprognosis of disease, as well as in the monitoring of diseaseprogression.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the CD200 antigen on a cell(such as, e.g., an immune cell), the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello, Nature, 305:537-539(1983)). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulinheavy-chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. For further details of illustrative currentlyknown methods for generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96/27011;Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med.175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553(1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993); Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J.Immunol. 147:60 (1991). Bispecific antibodies also include cross-linkedor heteroconjugate antibodies. Heteroconjugate antibodies may be madeusing any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins may be linkedto the Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers may be reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).Alternatively, the antibodies can be “linear antibodies” as described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)—C_(H)1—V_(H)—C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

III. Methods of Treating Patients with Autoimmune Disorders

In certain aspects, the disclosure relates to treating patients withautoimmune disorders with a therapy comprising an anti-CD200 antibody orantigen-binding fragment thereof. In certain aspects, the disclosurerelates to treating patients with an unwanted immune response with atherapy comprising an anti-CD200 antibody or antigen-binding fragmentthereof. The antibody may be antagonistic, agonistic or a non-blockingantibody and may be a murine, chimeric, humanized, human or de-immunizedanti-CD200 antibody. Thus, methods of treating patients with autoimmunedisorders or an unwanted immune response may comprise any of the CD200antibodies as set forth in the present disclosure.

In certain embodiments, anti-CD200 antibodies may be used for depletingany type of cell that expresses CD200 on its surface, including forexample, immune cells such as T-cells, B-cells, and dendritic cells. Inone embodiment, anti-CD200 antibodies may be useful for targeteddestruction of immune cells involved in an unwanted immune response.

In certain aspects, the disclosure relates to treating patients withautoimmune disorders or an unwanted immune response with a therapycomprising an anti-CD200 antibody or antigen-binding fragment thereofthat blocks the production of auto-antibodies. In certain embodiments,said auto-antibodies are selected from IgG1, IgG2, IgG3, IgG4, IgM,IgA1, IgA2, IgA, IgD, and/or IgE immunoglobulins. In certainembodiments, said antibody may be any antibody or antigen-bindingfragment thereof of the application. In certain embodiments, an antibodyor antigen-binding fragment thereof of the application does not blockthe production of auto-antibodies.

In certain aspects, the disclosure relates to treating patients withautoimmune disorders or an unwanted immune response with a therapycomprising an anti-CD200 antibody or antigen-binding fragment thereofthat modulates expression of cytokines in said patient. In certainembodiments, said antibody or antigen-binding fragment thereof enhancesproduction of a cytokine in said patient selected from the groupconsisting of: IL-12, IL-10 and IL-4. In certain embodiments, saidantibody or antigen-binding fragment thereof modulates production of acytokine in said patient selected from the group consisting of: IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,and IL-33. In certain embodiments, said antibody may be any antibody orantigen-binding fragment thereof of the application.

An unwanted immune response may be, for example, immune responsesassociated with an autoimmune disorder, transplants, allergies, orinflammatory disorders. Exemplary autoimmune diseases and disorders thatmay be treated with the anti-CD200 antibodies provided herein include,for example, inflammatory responses such as inflammatory skin diseasesincluding psoriasis and dermatitis (e.g. atopic dermatitis);dermatomyositis; systemic scleroderma and sclerosis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis); respiratory distress syndrome (including adultrespiratory distress syndrome; ARDS); dermatitis; meningitis;encephalitis; uveitis; colitis; glomerulonephritis; allergic conditionssuch as eczema and asthma and other conditions involving infiltration ofT cells and chronic inflammatory responses; atherosclerosis; leukocyteadhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus(SLE); diabetes mellitus (e.g. Type I diabetes mellitus or insulindependent diabetes mellitus); multiple sclerosis; Reynaud's syndrome;autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome;juvenile onset diabetes; and immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytestypically found in tuberculosis, sarcoidosis, polymyositis,granulomatosis and vasculitis; pernicious anemia (Addison's disease);diseases involving leukocyte diapedesis; central nervous system (CNS)inflammatory disorder; multiple organ injury syndrome; hemolytic anemia(including, but not limited to cryoglobinemia or Coombs positiveanemia); myasthenia gravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter'sdisease; stiff-man syndrome; Bechet disease; giant cell arteritis;immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia andautoimmune hemolytic diseases, Hashimoto's thyroiditis, Wegener'sgranulomatosis, cold agglutinin disease associated with indolentlymphoma, acquired factor VIII inhibitors disease, etc.

Therapies comprising CD200 antibodies may be administered to patients incombination therapies. Accordingly, targeted killing of certainpopulations of immune cells for treating or preventing autoimmunedisorders, enhancing or extending transplant survival, treating orpreventing allergies, or treating or preventing inflammatory disorders,may be administered as part of a combination therapy. For example, apatient receiving a first therapy comprising a CD200 antibody (e.g., ananti-CD200 antibody described herein) may also be given a secondtherapy. The CD200 antibody may be given simultaneously with the secondtherapy. Alternatively, the CD200 antibody may be given prior to orfollowing the second therapy. Second therapies include but are notlimited to anti-inflammatory agents, immunosuppressive agents, and/oranti-infective agents.

Combination therapies of the present disclosure include, for example, aCD200 antibody as described herein administered concurrently orsequentially in series with steroids, anti-malarials, aspirin,non-steroidal anti-inflammatory drugs, immunosuppressants, or cytotoxicdrugs. Included are corticosteroids (e.g. prednisone, dexamethasone, andprednisolone), methotrexate, methylprednisolone, macrolideimmunosuppressants (e.g. sirolimus and tacrolimus), mitotic inhibitors(e.g. azathioprine, cyclophosphamide, and methotrexate), fungalmetabolites that inhibit the activity of T lymphocytes (e.g.cyclosporine), mycophenolate mofetil, glatiramer acetate, and cytotoxicand DNA-damaging agents (e.g. chlorambucil). For autoimmune disordersanti-CD200 therapy may be combined with antibody treatments includingdaclizumab, a genetically engineered human IgG1 monoclonal antibody thatbinds specifically to the α-chain of the interleukin-2 receptor, as wellas various other antibodies targeting immune cells or other cells. Suchcombination therapies may be useful in the treatment of type 1 diabetes,rheumatoid arthritis, lupus, and idiopathic thrombocytopenic purpura,and other autoimmune indications. The disclosure also relates totherapies for autoimmune disorders and for transplant patientscomprising a CD200 antibody (such as, for example, the antibodies andvariants thereof described in the present disclosure) conjugated to oneor more agent.

IV. Modes of Administration and Formulations

The route of antibody administration of the antibodies of the presentdisclosure (whether the pure antibody, a labeled antibody, an antibodyfused to a toxin, etc.) is in accord with known methods, e.g., injectionor infusion by intravenous, intraperitoneal, intracerebral,intramuscular, subcutaneous, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, or by sustained release systems. Theantibody is preferably administered continuously by infusion or by bolusinjection. One may administer the antibodies in a local or systemicmanner.

The present antibodies may be prepared in a mixture with apharmaceutically acceptable carrier. Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition. This therapeutic composition can be administeredintravenously or through the nose or lung, preferably as a liquid orpowder aerosol (lyophilized). The composition may also be administeredparenterally or subcutaneously as desired. When administeredsystemically, the therapeutic composition should be sterile,substantially pyrogen-free and in a parenterally acceptable solutionhaving due regard for pH, isotonicity, and stability. For example, apharmaceutical preparation is substantially free of pyrogenic materialsso as to be suitable for administration as a human therapeutic. Theseconditions are known to those skilled in the art.

In certain embodiments, any antibody or antigen-binding fragment thereofof the application is administered acutely to said mammal. In certainembodiments, said antibody or antigen-binding fragment thereof isadministered for at least one month to said mammal. In certainembodiments, said antibody or antigen-binding fragment thereof isadministered for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months tosaid mammal. In certain embodiments, said antibody or antigen-bindingfragment thereof is administered for at least one year to said mammal.In certain embodiments, said antibody or antigen-binding fragmentthereof is administered for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 yearsto said mammal. In certain embodiments, said antibody or antigen-bindingfragment thereof is administered chronically to said mammal, i.e.,recurrently for at least 14 days, 28 days, 3 months, 6 months, 1 year, 5years, or longer. In certain embodiments, said antibody orantigen-binding fragment thereof is administered to said mammal for theremainder of its life.

Pharmaceutical compositions suitable for use include compositionswherein one or more of the present antibodies are contained in an amounteffective to achieve their intended purpose. More specifically, atherapeutically effective amount means an amount of antibody effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein. Therapeutically effective dosages may be determined byusing in vitro and in vivo methods.

EXEMPLIFICATION Example 1 Materials and Methods Induction and Evaluationof Collagen Induced Arthritis (CIA) Preparation of Reagents:

1. Preparation of BCII: (Bovine type II Collagen from Elastin Products).BCII is reconstituted by stirring overnight in cold room in 0.01 MAcetic Acid at a concentration of 4 mg/mL.

2. Preparation of Complete Freund's Adjuvant (CFA) H37Ra (Difco):

10 mg Mycobacterium tuberculosis is added to 10 ml Complete Freund'sAdjuvant which contains 10 mg Mycobacterium tuberculosis so that thefinal concentration for Mycobacterium tuberculosis is 2 mg/mL. CFA isstirred overnight at 4° C. overnight.3. Preparation of emulsion (4° C.):1:1 ratio of 4 mg/mL BCII and CFA with Mycobacterium tuberculosis (2mg/mL) 100 μL emulsion=200 μg BCII+100 μg CFA-M.T H37Ra4. Intradermal injection:Inject 150 μL of emulsion intradermally at the base of the mouse tail.Mice will be re-immunized 21 days after the first immunization followingthe identical protocol.

Evaluation of CIA:

The severity of arthritis was determined by scoring and measuring thefront paws, hind paws, elbow and knee joints with a caliper.

-   -   1. Arthritis scores:        -   0=No paw swelling        -   1=mild/moderate visible erythema and swelling        -   2=severe erythema and swelling affecting an entire paw or            joint        -   3=deformed paw or joint with ankylosis

The parameters for determining the above scores were:

Paw Elbow Knee 0 - Normal 0 - <3.3 mm 0 - <4 mm 1 - R/SW < 2.2 mm 1 - SW3.4-3.5 mm 1 - SW 4.3-4.5 mm 2 - R/SW 2.2-3.2 mm 2 - R/SW 3.5-3.6 mm 2 -R/SW 4.5-5 mm 3 - R/SW > 3.2 mm 3 - R/SW > 3.6 mm 3 - R/SW > 5 mmR/SW—redness and swelling

-   -   2. The degree of swelling was visually examined and measured        with a caliper at the following time points:        -   Time: 1) before immunization.            -   2) once/day starting from day 21 to day 42.                A Total Arthritic Score was calculated by adding the                measurements for each paw, elbow and knee (total of 8                measurements per mouse thus yielding a maximal score of                24).

Serum Collection Time:

-   -   1. Before immunization    -   2. day 14 and day 28 for prevention treatment group, day 31 for        therapeutic treatment group.    -   3. day 42 after first immunization    -   4. Serum anti-collagen antibodies (B cell response) were        measured at the times indicated in FIGS. 2 and 3B.    -   5. Spleen cells for cytokine measurement were taken when the        animals were sacrificed.        Histological Examination: Day 42 after First Immunization

Protocol for Intracellular Staining for Cytokine Detection (Splenocytes)

1. Collect animal spleen in plain HBSS or PBS on ice;2. Homogenize the spleen to collect splenocytes in 5-10 mL of plain HBSSor PBS, spin down the cells by 1,250 rpm×5 min, rm. tp.;3. Discard supernatant, re-suspend the cell pellet by vortex, add 5-10mL of ACK cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA2H₂O) on to the cells for 3 min. at rm. tp.;4. Add FACS Washing/Staining Buffer (2% FBS/HBSS+0.02% Sodium Azide)fill to the top of the tube and spin down the cells by 1,250 rpm×5 min,rm. tp.;5. Repeat wash twice;6. Count the cells and then distribute 1.0×10⁶ cells/50 μL/well in tothe 96 well U-bottom plate;7. Add antibodies (0.1 μg/well=1.0×10⁶ cells) into the wells accordingto the staining plan, if both antibodies for the double staining are forthe cell surface markers these can be added together in this step, for30-60 min. 4° C. in dark;8. Repeat wash 3 times using FACS washing/staining buffer, 250 μL/well;9. Add CytoFix buffer (BD Pharmingen Kit) 250 μL/well for 30 min. 4° C.in dark;10. Wash wells with CytoPerm/CytoWash 3 times;11. Re-suspend cells with 50 μL/well of CytoPerm/CytoWash buffer, addantibodies against cytokines (0.1 μg/well=1.0×10⁶ cells), incubate for30-60 min. 4° C. in dark;12. Repeat wash with FACS washing buffer twice, see above #8, wash plateonce with plain PBS;Re-suspend wells to add 250 μL/well of plain PBS, transfer the cellsinto the FACS tube. The samples are now ready for running the FACS.

Example 2 Evaluation of Anti-CD200 on Arthritis Animal Model

Administration of anti-CD200 antibody was performed in mice to test: 1)whether administration of anti-CD200 antibody prevents the developmentof arthritis and 2) whether administration of anti-CD200 antibodyreduces the severity of existing arthritis. A collagen induced arthritis(CIA) mouse model was used (mouse strain: DBA/1LacJ from Jackson Labs,male, 8 to 12 weeks old).

The anti-CD200 mAb used was OX90mG2a, a chimeric antibody derived fromOX90, a rat anti-mouse CD200 mAb obtained as a hybridoma from theEuropean Collection of Cell Cultures (ECACC No. 03062502; see Hoek etal., Science 290:1768-1771 (2000)). The rat antibody was geneticallymodified to contain the rat heavy chain variable regions fused to amurine IgG2a constant region and the rat light chain variable regionfused to a murine kappa constant region. An isotype matched control mAb,r12B4 was used as a control.

A) Prevention of Arthritis

Ten DBA/1LacJ mice were administered a 5 mg/kg dose of either anti-CD200or isotype-matched control mAb by i.p. injection from day 1 to day 7 andday 21 to day 25 after initial BCII immunization on day 1. An additional10 mice were treated again at day 21 to day 25 and were terminated atday 42. Mice were bled at day 14 to measure antibody response.

As seen in FIG. 1, anti-CD200 treatment reduces the severity of collageninduced arthritis (FIG. 1). Anti-CD200 treatment also inhibits theproduction of anti-collagen antibody production (FIG. 2). Serum levelsand subtypes of anti-BCII Abs were evaluated for the indicated treatmentgroups. DBA/1LacJ mice were i.p. injected with either anti-CD200 orisotype-matched control mAb from day 1 to day 7 and day 21 to day 25 andbled at pre-immunization, day 14, day 28 and day 42 to day 45 afterinitial BCII immunization on day 1.

B) Amelioration of Established Arthritis

DBA/1LacJ mice were administered a 5 mg/kg dose of either anti-CD200 orisotype-matched control mAb by i.p. injection on day 21 to day 30 afterinitial BCII immunization on day 1. Serum levels and subtypes ofanti-BCII Abs were evaluated for the indicated treatment groups. DBA/1LacJ mice were i.p. injected with either anti-CD200 or isotype-matchedcontrol mAb from day 21 to day 30 and bled at pre-immunization, day 14,day 28 and day 42 to day 45 after initial BCII immunization on day 1. Asshown in FIGS. 3A-B, anti-CD200 treatment can ameliorate establishedjoint inflammation independently of the effect on autoantibodyproduction.

Anti-CD200 treatment affects splenic cytokine profiles when administeredat various time points relative to collagen immunization of DBA/1 mice(FIGS. 4A-4B). Spleen cells were isolated from BCII immunized DBA/1LacJmice, which were treated with either anti-CD200 or isotype-matchedcontrol mAb from day 1 to day 7 and day 21 to day25 after initial BCIIimmunization on day 1. The percentage of IL-4, IL-10, TNF-α and INF-γproducing cells were analyzed by intracellular staining with anti-IL-4,anti-IL-10, anti-TNF-α, anti-INF-γ or with isotype-matched control IgG1Ab. Spleen cells were isolated from BCII immunized DBA/1LadJ mice, whichwere treated with either anti-CD200 or isotype-matched control mAb fromday 21 to day30 after initial BCII immunization on day 1. The percentageof IL-4, IL-10, TNF-α and INF-γ producing cells were analyzed byintracellular staining with anti-IL-4, anti-IL-10, anti-TNF-α,anti-INF-γ or with isotype-matched control IgG1 Ab.

The effect of alteration of cytokine profile after anti-CD200 treatmentwas further demonstrated in an allogenic immune response, where BALB/cmice were immunized with C57B/c spleen cells (FIG. 5).

It will be understood that various modifications may be made to theembodiments disclosed herein. For example, as those skilled in the artwill appreciate, the specific sequences described herein can be alteredslightly without necessarily adversely affecting the functionality ofthe polypeptide, antibody or antibody fragment used in bindingOX-2/CD200. For instance, substitutions of single or multiple aminoacids in the antibody sequence can frequently be made without destroyingthe functionality of the antibody or fragment. Thus, it should beunderstood that polypeptides or antibodies having a degree of identitygreater than 70% to the specific antibodies described herein are withinthe scope of this disclosure. In particularly useful embodiments,antibodies having an identity greater than about 80% to the specificantibodies described herein are contemplated. In other usefulembodiments, antibodies having an identity greater than about 90% to thespecific antibodies described herein are contemplated. Therefore, theabove description should not be construed as limiting, but merely asexemplifications of preferred embodiments. Those skilled in the art willenvision other modifications within the scope and spirit of thisdisclosure.

REFERENCES

The following references are incorporated herein by reference to morefully describe the state of the art to which the present inventionpertains. Any inconsistency between these publications below or thoseincorporated by reference above and the present disclosure shall beresolved in favor of the present disclosure.

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1. A method for treating a patient who has an autoimmune or inflammatorydisease, said method comprising administering a therapeuticallyeffective amount of an agent which inhibits interaction between CD200and CD200R to said patient, wherein said autoimmune or inflammatorydisease is ameliorated.
 2. The method of claim 1, wherein saidautoimmune or inflammatory disease is selected from the group consistingof rheumatoid arthritis, inflammatory bowel disease, systemic lupuserythematosus, multiple sclerosis, Hashimoto's thyroiditis, perniciousanemia, Addison's disease, type I diabetes, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome,idiopathic thrombocytopenic purpura, hemolytic anemia, Wegener'sgranulomatosis, refractory dermatomyositis, cold agglutinin diseaseassociated with indolent lymphoma, acquired factor VIII inhibitorsdisease and Grave's disease.
 3. The method of claim 1, wherein saidagent reduces the production of auto-antibodies.
 4. The method of claim1, wherein said agent is an anti-CD200 antibody or antigen-bindingfragment thereof.
 5. The method claim 4, wherein said antibody orantibody fragment thereof is selected from the group consisting of apolyclonal antibody, a monoclonal antibody or antibody fragment, arecombinant antibody, a diabody, a chimerized or chimeric antibody orantibody fragment, a humanized antibody or antibody fragment, adeimmunized human antibody or antibody fragment, a fully human antibodyor antibody fragment, a single chain antibody, an Fv, an Fd, an Fab, anFab′, and an F(ab′)₂.
 6. The method of claim 4, wherein said antibody isa monoclonal antibody.
 7. The method of claim 1, wherein said agent haseffector function.
 8. The method of claim 1, wherein said agent lackseffector function.
 9. The method of claim 1, wherein said agent isadministered for at least one month to said mammal.
 10. The method ofclaim 1, wherein said agent is administered for at least one year tosaid mammal.
 11. The method of claim 1, wherein said agent isadministered chronically to said mammal.
 12. The method of claim 1,wherein said agent is administered systemically to said mammal.
 13. Themethod of claim 1, wherein said agent is administered locally to saidmammal.
 14. The method of claim 1, further comprising administering asecond agent or therapy.
 15. The method of claim 14, wherein said secondagent is administered either sequentially or simultaneously.
 16. Themethod of claim 1, wherein said immune response is a primary immuneresponse.
 17. The method of claim 1, wherein said immune response is asecondary immune response.